Biological Indicators A special type of living microbiological products that can be used to confirm the performance of sterilization equipment, validation of sterilization procedures, and monitoring of sterilization in production processes. Biological indicators used in sterilization validation are generally spores of bacteria.
A. Basic requirements for the preparation of biological indicators for microorganisms
Different sterilization methods The use of different biological indicators, the preparation of biological indicators of the selected microorganisms must have the following characteristics:
(1) the tolerability of the strain should be greater than the tolerability of all possible contaminating bacteria in the product to be sterilized.
(2) bacteria should be pathogenic.
(3) strains should be stable. Long life, easy to save.
(4) easy to cultivate. If the use of dormant spores, biological indicator dormant spore content to 90% or more.
b. Preparation of biological indicator
The preparation of the biological indicator should be carried out according to certain procedures. Before the preparation, the characteristics of the microorganism used, such as the D value (the heat resistance parameter of the microorganism, refer to the time required for the microorganism to kill 90% at a certain temperature, In minutes). The strain was cultured in a suitable medium. The culture should be made into a suspension in which the number of spores should be dominant and the spores should be suspended in an untreated liquid for preservation.
The biological indicator contains a certain amount of one or more spores. Can be made in various forms. Usually a certain number of spores attached to the inert carrier, such as filter paper, glass, stainless steel, plastic products, etc.: spore suspension can also be sealed in the ampoule; some biological indicators also equipped with a medium system. D value in addition to sterilization conditions, but also with the existence of microbial environment. Therefore, after a certain form of biological indicator preparation is completed, the D value and the total number of spores should be determined. Biological indicator should use the appropriate material packaging, and set the validity period. The carrier and packaging material should also ensure that the sterilant penetrates and is in full contact with the biological indicator while protecting the biological indicator from contamination. The design of the carrier and the packaging principle is to facilitate storage, transportation, sampling, transfer inoculation.
Some biological indicators may be used to directly inoculate spores into liquid sterilants or substitutes with similar physical and chemical properties. When using substitutes, the application data proves the equivalence of the two.
C. Application of biological indicator
In the validation of the sterilization procedure, although the sterilization effect can be assessed by monitoring some of the parameters of the sterilization process, the degree of killing of the biological indicator is the most intuitive indicator of the effectiveness of a sterilization procedure. A commercially available standard biological indicator can also be used, and spores prepared from microorganisms with the most tolerant separation from the daily production of contaminating bacteria can also be used. In the biological indicator validation test, the D value of the spores under actual sterilization conditions was determined and the purity and number of spores were determined. In the case of validation, the amount of microbiological use of the biological indicator should be greater than the daily detection of microbial contamination, and the tolerance is strong to ensure greater safety of the sterilization procedure. In the final sterilization method, the biological indicator should be placed in different parts of the sterilization cabinet. And to avoid direct contact with the indicator to the sterilized article. The biological indicator is sterilized according to the set conditions and then cultured in the culture medium to determine whether the spores in the biological indicator are completely killed.
Excessive kill product sterilization verification generally does not take into account the level of microbial contamination, can be used commercially available biological indicator. For products that are poorly tolerated by sterilization methods, when the sterilization procedure is designed, the number of bacteria and spores of the biological indicator is selected based on the level of microbial contamination of the product in the production process. The sterility of such products should be assessed by monitoring the data obtained by verifying the amount of microbial contamination prior to each batch of sterilization, the tolerability and the validation of the sterilization procedure.
D. Commonly used biological indicator
(1) Damp-heat sterilization method The most commonly used bovine indicator is spores of Bacillus stearothermophilus, such as NCTC 10007, NCIMB 8157, ATCC 7953. D value of 1.5 ~ 3.0min, each piece (or per bottle) the number of active spores 5 × 105 ~ 5 × 106, at 121 ℃, 19min should be completely killed. In addition, Spores of Clostridium sporogenes (e.g., NCTC 8594, NCIMB 8053, ATCC 7955) and D values of 0.4 to 0.8 min can also be used.
(2) Dry heat sterilization The most common biological indicator for dry heat sterilization is the Spores of Bacillus subtilis, such as NCIMB 8058, ATCC 9372. D value is greater than 1.5min, the number of active spores per slice 5 × 105 ~ 5 × 106. Escherichia coli endomycin (Escherichia coliendomn), the amount of not less than 1000 bacterial endotoxin units.
(3) Radiation Sterilization The most commonly used biological indicator for radiation sterilization is Spores of Bacillus pumilus, such as NCTC 10327, NCIMB 10692, ATCC 27142. Each piece of live pieces 107 ~ 108, placed in the radiation dose 25kGy conditions, D value of about 3kGy. It should be noted that the microbes that are loaded in the sterilized article may exhibit stronger resistance to radiation than the Bacillus sp. Therefore, Bacillus sp. Can be used to monitor the sterilization process, but can not be used for the establishment of sterile radiation dose basis.
(4) Gas sterilization The most commonly used biological indicator for the sterilization of ethylene oxide is Spores of Bacillus subtilis (such as NCTC 10073, ATCC 9372). The most commonly used biological indicator for gaseous hydrogen peroxide sterilization is spores of Bacillus steatothermophilus, such as NCTC 10007, NCIMB 8157, ATCC 7953. The number of active spores per 1 × 106 ~ 5 × 106. Ethylene oxide sterilization, Bacillus subtilis spore D value greater than 2.5min, in the ethylene oxide concentration of 600mg / L, relative humidity of 60%, the temperature of 54 ℃ sterilization, 60min should be killed.
(5) Filter sterilization method The most commonly used biological indicator for the sterilization method is Pseudomonas diminuta (Pseudomonas diminuta, such as ATCC 19146), a filter for the membrane pore size of 0.22 μm; Serratia Marcescens) (ATCC 1 4756) for filters with a membrane pore size of 0.45 μm.